Mutations in human immunodeficiency virus type 1 (HIV-1) that confer drug resistance may also impair replication capacity of the virus. Reduced replication capacity of multi-drug resistant viruses may explain, in part, the persistent immunologic benefits of protease inhibitor-containing regimens in the setting of virologic failure. Plasma virus titers remain significantly below the viral set point due to the combined effects of the residual activity of the failing regimen and the reduced replication capacity compared to wild-type. Removal of selective pressure by treatment interruption leads to re-emergence of the fitter wild-type virus that is associated with a rise in virus load and fall in CD4 cell count. Enfuvirtide (T-20) is a novel HIV- 1 entry inhibitor with potent activity in vitro and in vivo. This 36-amino acid peptide blocks HIV-1 entry by binding to the first heptad repeat (HR-1) of the gp41 ectodomain, thereby preventing formation of a hairpin loop that is essential for virus-cell fusion. Mutations at several positions in HR-1 confer resistance to T-20. Although resistance to T-20 can develop quickly, data from clinical studies suggest that T-20 remains at least partially active despite emergence of T-20-resistant virus. Preliminary data show that these viruses are less fit than wild-type in growth competition assays in the absence of drug using a novel recombinant marker virus assay developed in our laboratory. We therefore propose a series of experiments to characterize further the effects of T-20 resistance mutations on HIV-1 fitness, kinetics of HIV-1 entry, and virulence. Specific aims of these experiments are: 1) To compare fitness of T-20 -resistant viruses in presence and absence of drug; 2) To test the hypothesis that fitness loss associated with T-20 resistance is significantly correlated with persistent antiviral activity of T-20; and 3) To test the hypothesis that fitness differences associated with T-20 resistance mutations are due to differences in the rate of virus-cell fusion. Results of these studies will provide a deeper understanding of the molecular, virologic, and clinical consequences ofT-20 resistance, and may help guide the use of T-20 in salvage therapy.